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        Download the raw data used to create the plots in this report below:

        Note that additional data was saved in multiqc_data_1 when this report was generated.


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        If you use plots from MultiQC in a publication or presentation, please cite:

        MultiQC: Summarize analysis results for multiple tools and samples in a single report
        Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
        Bioinformatics (2016)
        doi: 10.1093/bioinformatics/btw354
        PMID: 27312411

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        About MultiQC

        This report was generated using MultiQC, version 1.9.dev0

        You can see a YouTube video describing how to use MultiQC reports here: https://youtu.be/qPbIlO_KWN0

        For more information about MultiQC, including other videos and extensive documentation, please visit http://multiqc.info

        You can report bugs, suggest improvements and find the source code for MultiQC on GitHub: https://github.com/ewels/MultiQC

        MultiQC is published in Bioinformatics:

        MultiQC: Summarize analysis results for multiple tools and samples in a single report
        Philip Ewels, Måns Magnusson, Sverker Lundin and Max Käller
        Bioinformatics (2016)
        doi: 10.1093/bioinformatics/btw354
        PMID: 27312411

        A modular tool to aggregate results from bioinformatics analyses across many samples into a single report.

        Report generated on 2019-11-28, 14:47 based on data in: /project/RDS-FMH-Brainomics_Temp-RW/ad_rna


        General Statistics

        Showing 127/127 rows and 11/13 columns.
        Sample Name% Alignable% Proper PairsFrag Length% AlignedM Aligned% AlignedM Aligned% Dropped% Dups% GCM Seqs
        63
        217.6bp
        43.1%
        22.0
        PREC_S11_15
        100.0%
        91.4%
        38.5
        PREC_S11_15.R1
        2.8%
        39.6%
        46%
        48.4
        PREC_S11_15.R2
        34.3%
        47%
        48.4
        PREC_S11_15_R1_trim
        37.3%
        46%
        42.1
        PREC_S11_15_R2_trim
        32.7%
        46%
        42.1
        PREC_S11_15bam_stat
        99.9%
        PREC_S12_16
        100.0%
        92.2%
        43.2
        PREC_S12_16.R1
        2.8%
        35.9%
        45%
        54.3
        PREC_S12_16.R2
        29.5%
        45%
        54.3
        PREC_S12_16_R1_trim
        33.6%
        45%
        46.8
        PREC_S12_16_R2_trim
        27.9%
        45%
        46.8
        PREC_S12_16bam_stat
        99.9%
        PREC_S18_20
        100.0%
        91.2%
        36.9
        PREC_S18_20.R1
        2.6%
        37.6%
        46%
        46.5
        PREC_S18_20.R2
        32.3%
        46%
        46.5
        PREC_S18_20_R1_trim
        35.2%
        46%
        40.4
        PREC_S18_20_R2_trim
        30.8%
        46%
        40.4
        PREC_S18_20bam_stat
        99.9%
        PREC_S20_18
        100.0%
        90.3%
        40.9
        PREC_S20_18.R1
        2.7%
        42.8%
        47%
        52.2
        PREC_S20_18.R2
        37.8%
        47%
        52.2
        PREC_S20_18_R1_trim
        40.6%
        47%
        45.3
        PREC_S20_18_R2_trim
        36.4%
        47%
        45.3
        PREC_S20_18bam_stat
        99.9%
        PREC_S26_14
        100.0%
        88.2%
        32.8
        PREC_S26_14.R1
        3.1%
        43.1%
        50%
        44.1
        PREC_S26_14.R2
        38.5%
        50%
        44.1
        PREC_S26_14_R1_trim
        40.3%
        50%
        37.2
        PREC_S26_14_R2_trim
        36.8%
        49%
        37.2
        PREC_S26_14bam_stat
        99.8%
        PREC_S28_12
        100.0%
        90.6%
        38.5
        PREC_S28_12.R1
        2.8%
        38.3%
        47%
        49.2
        PREC_S28_12.R2
        33.4%
        47%
        49.2
        PREC_S28_12_R1_trim
        36.0%
        47%
        42.4
        PREC_S28_12_R2_trim
        31.8%
        47%
        42.4
        PREC_S28_12bam_stat
        99.9%
        PREC_S36_13
        100.0%
        91.2%
        40.8
        PREC_S36_13.R1
        2.9%
        46.4%
        48%
        52.1
        PREC_S36_13.R2
        40.8%
        49%
        52.1
        PREC_S36_13_R1_trim
        44.0%
        48%
        44.7
        PREC_S36_13_R2_trim
        39.4%
        48%
        44.7
        PREC_S36_13bam_stat
        99.9%
        PREC_S43_19
        100.0%
        92.6%
        47.6
        PREC_S43_19.R1
        2.7%
        38.8%
        44%
        58.9
        PREC_S43_19.R2
        32.6%
        45%
        58.9
        PREC_S43_19_R1_trim
        36.5%
        44%
        51.4
        PREC_S43_19_R2_trim
        31.0%
        44%
        51.4
        PREC_S43_19bam_stat
        99.9%
        PREC_S44_17
        100.0%
        92.0%
        44.8
        PREC_S44_17.R1
        2.8%
        40.0%
        46%
        56.4
        PREC_S44_17.R2
        34.2%
        46%
        56.4
        PREC_S44_17_R1_trim
        37.5%
        46%
        48.7
        PREC_S44_17_R2_trim
        32.6%
        46%
        48.7
        PREC_S44_17bam_stat
        99.9%
        PREC_S47_11
        100.0%
        91.5%
        34.3
        PREC_S47_11.R1
        2.7%
        36.8%
        45%
        43.3
        PREC_S47_11.R2
        30.8%
        46%
        43.3
        PREC_S47_11_R1_trim
        34.5%
        45%
        37.5
        PREC_S47_11_R2_trim
        29.2%
        45%
        37.5
        PREC_S47_11bam_stat
        99.9%
        SHSY5Y_CTRL_21
        100.0%
        91.8%
        1.8
        SHSY5Y_CTRL_21.R1
        3.4%
        9.7%
        42%
        2.3
        SHSY5Y_CTRL_21.R2
        7.8%
        43%
        2.3
        SHSY5Y_CTRL_21_R1_trim
        8.6%
        42%
        1.9
        SHSY5Y_CTRL_21_R2_trim
        7.0%
        42%
        1.9
        SHSY5Y_CTRL_21bam_stat
        99.9%
        VIC_S11_5
        100.0%
        89.1%
        28.3
        VIC_S11_5.R1
        2.8%
        31.4%
        44%
        36.7
        VIC_S11_5.R2
        24.5%
        44%
        36.7
        VIC_S11_5_R1_trim
        29.4%
        44%
        31.7
        VIC_S11_5_R2_trim
        23.2%
        44%
        31.7
        VIC_S11_5bam_stat
        99.7%
        VIC_S12_6
        100.0%
        87.0%
        44.8
        VIC_S12_6.R1
        2.9%
        41.9%
        46%
        59.4
        VIC_S12_6.R2
        34.6%
        46%
        59.4
        VIC_S12_6_R1_trim
        39.3%
        46%
        51.5
        VIC_S12_6_R2_trim
        33.0%
        46%
        51.5
        VIC_S12_6bam_stat
        99.6%
        VIC_S18_10
        100.0%
        90.2%
        38.5
        VIC_S18_10.R1
        2.7%
        34.8%
        45%
        49.1
        VIC_S18_10.R2
        28.3%
        45%
        49.1
        VIC_S18_10_R1_trim
        32.7%
        45%
        42.7
        VIC_S18_10_R2_trim
        26.9%
        45%
        42.7
        VIC_S18_10bam_stat
        99.8%
        VIC_S20_8
        100.0%
        89.9%
        39.3
        VIC_S20_8.R1
        3.0%
        39.9%
        46%
        51.0
        VIC_S20_8.R2
        33.7%
        46%
        51.0
        VIC_S20_8_R1_trim
        37.3%
        45%
        43.7
        VIC_S20_8_R2_trim
        32.1%
        45%
        43.7
        VIC_S20_8bam_stat
        99.8%
        VIC_S26_4
        100.0%
        88.5%
        43.3
        VIC_S26_4.R1
        2.9%
        36.8%
        47%
        57.3
        VIC_S26_4.R2
        30.5%
        47%
        57.3
        VIC_S26_4_R1_trim
        34.2%
        47%
        48.9
        VIC_S26_4_R2_trim
        29.1%
        47%
        48.9
        VIC_S26_4bam_stat
        99.7%
        VIC_S28_2
        100.0%
        89.3%
        54.0
        VIC_S28_2.R1
        2.8%
        39.4%
        45%
        69.8
        VIC_S28_2.R2
        33.3%
        45%
        69.8
        VIC_S28_2_R1_trim
        36.9%
        45%
        60.5
        VIC_S28_2_R2_trim
        31.8%
        45%
        60.5
        VIC_S28_2bam_stat
        99.7%
        VIC_S36_3
        100.0%
        90.8%
        49.0
        VIC_S36_3.R1
        2.9%
        40.3%
        45%
        62.5
        VIC_S36_3.R2
        33.1%
        45%
        62.5
        VIC_S36_3_R1_trim
        38.0%
        45%
        54.0
        VIC_S36_3_R2_trim
        31.6%
        45%
        54.0
        VIC_S36_3bam_stat
        99.8%
        VIC_S43_9
        100.0%
        90.2%
        43.5
        VIC_S43_9.R1
        2.7%
        39.6%
        46%
        56.3
        VIC_S43_9.R2
        33.3%
        46%
        56.3
        VIC_S43_9_R1_trim
        37.2%
        45%
        48.2
        VIC_S43_9_R2_trim
        31.7%
        45%
        48.2
        VIC_S43_9bam_stat
        99.8%
        VIC_S44_7
        100.0%
        90.3%
        46.1
        VIC_S44_7.R1
        2.7%
        38.6%
        45%
        59.0
        VIC_S44_7.R2
        31.8%
        45%
        59.0
        VIC_S44_7_R1_trim
        36.3%
        45%
        51.1
        VIC_S44_7_R2_trim
        30.2%
        45%
        51.1
        VIC_S44_7bam_stat
        99.8%
        VIC_S47_1
        100.0%
        89.5%
        48.5
        VIC_S47_1.R1
        2.7%
        37.2%
        44%
        62.3
        VIC_S47_1.R2
        30.0%
        44%
        62.3
        VIC_S47_1_R1_trim
        34.9%
        44%
        54.2
        VIC_S47_1_R2_trim
        28.5%
        44%
        54.2
        VIC_S47_1bam_stat
        99.7%

        Rsem

        Rsem RSEM (RNA-Seq by Expectation-Maximization) is a software package forestimating gene and isoform expression levels from RNA-Seq data.

        Mapped Reads

        A breakdown of how all reads were aligned for each sample.

        loading..

        Multimapping rates

        A frequency histogram showing how many reads were aligned to n reference regions.

        In an ideal world, every sequence reads would align uniquely to a single location in the reference. However, due to factors such as repeititve sequences, short reads and sequencing errors, reads can be align to the reference 0, 1 or more times. This plot shows the frequency of each factor of multimapping. Good samples should have the majority of reads aligning once.

        loading..

        RSeQC

        RSeQC package provides a number of useful modules that can comprehensively evaluate high throughput RNA-seq data.

        Read Distribution

        Read Distribution calculates how mapped reads are distributed over genome features.

        loading..

        Inner Distance

        Inner Distance calculates the inner distance (or insert size) between two paired RNA reads. Note that this can be negative if fragments overlap.

        loading..

        Read GC Content

        read_GC calculates a histogram of read GC content.

        loading..

        Read Duplication

        read_duplication.py calculates how many alignment positions have a certain number of exact duplicates. Note - plot truncated at 500 occurrences and binned.

        loading..

        Junction Annotation

        Junction annotation compares detected splice junctions to a reference gene model. An RNA read can be spliced 2 or more times, each time is called a splicing event.

           
        loading..

        Junction Saturation

        Junction Saturation counts the number of known splicing junctions that are observed in each dataset. If sequencing depth is sufficient, all (annotated) splice junctions should be rediscovered, resulting in a curve that reaches a plateau. Missing low abundance splice junctions can affect downstream analysis.

        Click a line to see the data side by side (as in the original RSeQC plot).

        loading..

        Infer experiment

        Infer experiment counts the percentage of reads and read pairs that match the strandedness of overlapping transcripts. It can be used to infer whether RNA-seq library preps are stranded (sense or antisense).

        loading..

        Bam Stat

        All numbers reported in millions.

        loading..

        Kallisto

        Kallisto is a program for quantifying abundances of transcripts from RNA-Seq data.

        loading..

        STAR

        STAR is an ultrafast universal RNA-seq aligner.

        Alignment Scores

        loading..

        Trimmomatic

        Trimmomatic is a flexible read trimming tool for Illumina NGS data.

        loading..

        FastQC

        FastQC is a quality control tool for high throughput sequence data, written by Simon Andrews at the Babraham Institute in Cambridge.

        Sequence Counts

        Sequence counts for each sample. Duplicate read counts are an estimate only.

        This plot show the total number of reads, broken down into unique and duplicate if possible (only more recent versions of FastQC give duplicate info).

        You can read more about duplicate calculation in the FastQC documentation. A small part has been copied here for convenience:

        Only sequences which first appear in the first 100,000 sequences in each file are analysed. This should be enough to get a good impression for the duplication levels in the whole file. Each sequence is tracked to the end of the file to give a representative count of the overall duplication level.

        The duplication detection requires an exact sequence match over the whole length of the sequence. Any reads over 75bp in length are truncated to 50bp for this analysis.

        loading..

        Sequence Quality Histograms

        The mean quality value across each base position in the read.

        To enable multiple samples to be plotted on the same graph, only the mean quality scores are plotted (unlike the box plots seen in FastQC reports).

        Taken from the FastQC help:

        The y-axis on the graph shows the quality scores. The higher the score, the better the base call. The background of the graph divides the y axis into very good quality calls (green), calls of reasonable quality (orange), and calls of poor quality (red). The quality of calls on most platforms will degrade as the run progresses, so it is common to see base calls falling into the orange area towards the end of a read.

        loading..

        Per Sequence Quality Scores

        The number of reads with average quality scores. Shows if a subset of reads has poor quality.

        From the FastQC help:

        The per sequence quality score report allows you to see if a subset of your sequences have universally low quality values. It is often the case that a subset of sequences will have universally poor quality, however these should represent only a small percentage of the total sequences.

        loading..

        Per Base Sequence Content

        The proportion of each base position for which each of the four normal DNA bases has been called.

        To enable multiple samples to be shown in a single plot, the base composition data is shown as a heatmap. The colours represent the balance between the four bases: an even distribution should give an even muddy brown colour. Hover over the plot to see the percentage of the four bases under the cursor.

        To see the data as a line plot, as in the original FastQC graph, click on a sample track.

        From the FastQC help:

        Per Base Sequence Content plots out the proportion of each base position in a file for which each of the four normal DNA bases has been called.

        In a random library you would expect that there would be little to no difference between the different bases of a sequence run, so the lines in this plot should run parallel with each other. The relative amount of each base should reflect the overall amount of these bases in your genome, but in any case they should not be hugely imbalanced from each other.

        It's worth noting that some types of library will always produce biased sequence composition, normally at the start of the read. Libraries produced by priming using random hexamers (including nearly all RNA-Seq libraries) and those which were fragmented using transposases inherit an intrinsic bias in the positions at which reads start. This bias does not concern an absolute sequence, but instead provides enrichement of a number of different K-mers at the 5' end of the reads. Whilst this is a true technical bias, it isn't something which can be corrected by trimming and in most cases doesn't seem to adversely affect the downstream analysis.

        Click a sample row to see a line plot for that dataset.
        Rollover for sample name
        Position: -
        %T: -
        %C: -
        %A: -
        %G: -

        Per Sequence GC Content

        The average GC content of reads. Normal random library typically have a roughly normal distribution of GC content.

        From the FastQC help:

        This module measures the GC content across the whole length of each sequence in a file and compares it to a modelled normal distribution of GC content.

        In a normal random library you would expect to see a roughly normal distribution of GC content where the central peak corresponds to the overall GC content of the underlying genome. Since we don't know the the GC content of the genome the modal GC content is calculated from the observed data and used to build a reference distribution.

        An unusually shaped distribution could indicate a contaminated library or some other kinds of biased subset. A normal distribution which is shifted indicates some systematic bias which is independent of base position. If there is a systematic bias which creates a shifted normal distribution then this won't be flagged as an error by the module since it doesn't know what your genome's GC content should be.

        loading..

        Per Base N Content

        The percentage of base calls at each position for which an N was called.

        From the FastQC help:

        If a sequencer is unable to make a base call with sufficient confidence then it will normally substitute an N rather than a conventional base call. This graph shows the percentage of base calls at each position for which an N was called.

        It's not unusual to see a very low proportion of Ns appearing in a sequence, especially nearer the end of a sequence. However, if this proportion rises above a few percent it suggests that the analysis pipeline was unable to interpret the data well enough to make valid base calls.

        loading..

        Sequence Length Distribution

        The distribution of fragment sizes (read lengths) found. See the FastQC help

        loading..

        Sequence Duplication Levels

        The relative level of duplication found for every sequence.

        From the FastQC Help:

        In a diverse library most sequences will occur only once in the final set. A low level of duplication may indicate a very high level of coverage of the target sequence, but a high level of duplication is more likely to indicate some kind of enrichment bias (eg PCR over amplification). This graph shows the degree of duplication for every sequence in a library: the relative number of sequences with different degrees of duplication.

        Only sequences which first appear in the first 100,000 sequences in each file are analysed. This should be enough to get a good impression for the duplication levels in the whole file. Each sequence is tracked to the end of the file to give a representative count of the overall duplication level.

        The duplication detection requires an exact sequence match over the whole length of the sequence. Any reads over 75bp in length are truncated to 50bp for this analysis.

        In a properly diverse library most sequences should fall into the far left of the plot in both the red and blue lines. A general level of enrichment, indicating broad oversequencing in the library will tend to flatten the lines, lowering the low end and generally raising other categories. More specific enrichments of subsets, or the presence of low complexity contaminants will tend to produce spikes towards the right of the plot.

        loading..

        Overrepresented sequences

        The total amount of overrepresented sequences found in each library.

        FastQC calculates and lists overrepresented sequences in FastQ files. It would not be possible to show this for all samples in a MultiQC report, so instead this plot shows the number of sequences categorized as over represented.

        Sometimes, a single sequence may account for a large number of reads in a dataset. To show this, the bars are split into two: the first shows the overrepresented reads that come from the single most common sequence. The second shows the total count from all remaining overrepresented sequences.

        From the FastQC Help:

        A normal high-throughput library will contain a diverse set of sequences, with no individual sequence making up a tiny fraction of the whole. Finding that a single sequence is very overrepresented in the set either means that it is highly biologically significant, or indicates that the library is contaminated, or not as diverse as you expected.

        FastQC lists all of the sequences which make up more than 0.1% of the total. To conserve memory only sequences which appear in the first 100,000 sequences are tracked to the end of the file. It is therefore possible that a sequence which is overrepresented but doesn't appear at the start of the file for some reason could be missed by this module.

        loading..

        Adapter Content

        The cumulative percentage count of the proportion of your library which has seen each of the adapter sequences at each position.

        Note that only samples with ≥ 0.1% adapter contamination are shown.

        There may be several lines per sample, as one is shown for each adapter detected in the file.

        From the FastQC Help:

        The plot shows a cumulative percentage count of the proportion of your library which has seen each of the adapter sequences at each position. Once a sequence has been seen in a read it is counted as being present right through to the end of the read so the percentages you see will only increase as the read length goes on.

        loading..

        Status Checks

        Status for each FastQC section showing whether results seem entirely normal (green), slightly abnormal (orange) or very unusual (red).

        FastQC assigns a status for each section of the report. These give a quick evaluation of whether the results of the analysis seem entirely normal (green), slightly abnormal (orange) or very unusual (red).

        It is important to stress that although the analysis results appear to give a pass/fail result, these evaluations must be taken in the context of what you expect from your library. A 'normal' sample as far as FastQC is concerned is random and diverse. Some experiments may be expected to produce libraries which are biased in particular ways. You should treat the summary evaluations therefore as pointers to where you should concentrate your attention and understand why your library may not look random and diverse.

        Specific guidance on how to interpret the output of each module can be found in the relevant report section, or in the FastQC help.

        In this heatmap, we summarise all of these into a single heatmap for a quick overview. Note that not all FastQC sections have plots in MultiQC reports, but all status checks are shown in this heatmap.

        loading..